Enzyme immobilization: fundamentals and application Xiaoli wang 2013.11.30
Dec 30, 2015
Enzyme immobilization:
fundamentals and application
Xiaoli wang2013.11.30
Enzyme ImmobilizationEnzyme Immobilization
• enhanced stability • ease of separation
Advantages
Disadvantages• lowered activity
• additional costs
Immobilization of enzymes on mesoporous silicate materials
Immobilization of enzymes on mesoporous silicate materials
Microporous (<2nm), mesoporous (2-50nm), macroporous (>50nm)
BET: adsorption–desorption isotherms obtained can be classified
into six types, I–VI.
TEM TEM can provide information on the size of pores and of the pore walls as well as the degree of short range and long range order of the material.
XRD
XRD can be used with materials containing ordered mesoporous channels to ascertain if the structure is hexagonal, cubic, lamellar or disordered.
Immobilization of enzymes on mesoporous silicate materials
Immobilization of enzymes on mesoporous silicate materials
Advantages of mesoporous materials
•ordered pore structures,
•narrow pore size distributions,
•high surface areas
•high stability and can be
•chemically modified with various functional groups.
Methods Enzymol., 1976, 44, 776.
controlled pore glass (CPG) controlled pore glass (CPG)
The first report on the immobilisation of enzymes on controlled
pore glass (CPG) was described by Weetall.
CPG with pore sizes ranging from 10 to 300 nm are commercially
available, and can be prepared with particle sizes of 100 mm, a size
suitable for use in packed bed reactors or columns.
A major disadvantage of the material was the surface area rapidly
decreases with increasing pore size.
Methods Enzymol., 1976, 44, 776.
Sol–gel materials Sol–gel materials
Sol–gels are formed via hydrolysis and condensation of a precursor
species such as Si(OC2H5)4, Enzyme encapsulation occurs by
placing the enzyme in the reaction mixture.
While the production of ethanol may be detrimental to the activity
of the enzyme.
Encapsulation introduces a diffusion barrier which can reduce the
rate of delivery of substrate to the enzyme and the rate of removal
of the product.
Anal. Chim. Acta, 2002, 461, 1–36.
Mesoporous silicates Mesoporous silicates
Zeolites are among the most commercially important porous
materials and in widespread use in a range of industrially important
processes.
The synthesis of mesoporous silicates (MPS) was first described in
1971. MPS are formed using surfactants, which act as structure
directing agents.
Principles and Practice of Heterogeneous Catalysis, VCH, Weinheim, 1997.
US Pat., 3556725, 1971.
Functionalised mesoporous materials
Functionalised mesoporous materials
In addition to pure silicate materials, a wide range of functionalised
MPS can be prepared.
Post-synthesis modificationDirect functionalisationIncorporation of bridged silesquioxanes
Immobilisation of enzymes on poroussilicate materials
Immobilisation of enzymes on poroussilicate materials
Adsorption
Adsorption of enzymes on to MPS supports is controlled by the
pore dimensions, surface charge and composition of the support
together with the size, surface charge distribution and
hydrophilic/hydrophobic nature of the enzyme. Covalent immobilisation of enzymes
Covalent attachment of the enzyme to the surface can obviate the
problem of leaching, increase the stability and enable reuse of
the immobilised enzyme.
Conclusions Conclusions
Mesoporous silicates are attractive materials for use as supports
of the enzymes immobilisation.
The synthesis of MPS is relatively straight forward and
produces materials with well defined and ordered pore structures,
high surface areas and good mechanical and chemical stability.
Surface functionalisation of MPS can be utilised to produce a
material that can be tailored to suit the immobilisation of a
particular enzyme.
Enzymatic reactions on immobilised substrates
Enzymatic reactions on immobilised substrates
1. Radioactive labelling
2. Fluorescence
3. Electrochemical detection
4. Mass spectrometry
5. Surface plasmon resonance (SPR)
6. Atomic force microscopy (AFM)
7. Quartz crystal microbalance (QCM)
The following analytical techniques were available to
monitor enzyme activity on surfaces.
Chem. Soc. Rev., 2013, 42, 6378--6405
Enzymatic reactions on immobilised substrates
Enzymatic reactions on immobilised substrates
Not all enzymes are suitable for reactions on solid
support, in particular where active sites are deeply buried
in the enzyme structure. So far, there are no general rules
that would allow predictions on activity on the surface.
However, certain classes of enzymes have emerged as
being very successful.
Chem. Soc. Rev., 2013, 42, 6378--6405
An overview of all successful enzymatic transformations on
surfaces is provided as followed.
DNA polymerase, Hydrolases, Transferases
Kinases, Phosphatases, Proteases
Acetylases, Methyltransferases, Ligases
Glycosyltransferases
Lipases
Other hydrolases
This review has tried to compile the most common
enzymatic reactions performed on surfaces and address issues
of surface material and analytical readout.Chem. Soc. Rev., 2013, 42, 6378--6405
Immobilisation and application of lipases in organic media
Immobilisation and application of lipases in organic media
The natural reaction of lipases is to hydrolyse ester bonds.
lipases are excellent examples of ‘‘promiscuous’’ enzymes,
catalysing reactions quite different from the normal ones.
Lipase-catalysed aldol addition, racemisation and epoxidation
constitute a few interesting examples.
Chem. Soc. Rev., 2013, 42, 6406--6436
Effects of reaction conditions on lipase-catalysed reactions
Effects of reaction conditions on lipase-catalysed reactions
Factors cause the differences in enzyme activity Method of enzyme preparation (immobilisation, etc.) The quantity of water The kind of solvent The pH
The catalytic activity of enzymes in organic solvents is sometimes several orders of magnitude lower than in water, making practical applications unattractive. However, the situation can be improved by choosing the appropriate conditions for biocatalysis in organic solvents.
Lipase immobilisation Lipase immobilisation
Lipases can be immobilised using most of the methods developed
for enzyme immobilisation in general.
The major types of
immobilisation:
Adsorption
Entrapment
Covalent coupling
Adsorption Adsorption
Adsorption on inorganic supports. Adsorption on mesoporous silica. Adsorption on organic polymers. Organic solvent rinsing as an alternative drying method. Protein-coated microcrystals.
Physical adsorption is the simplest method of enzyme
immobilisation. In the case of lipases, hydrophobic interaction is
most common, but ionic interactions with ionexchange materials,
etc., can also be useful.
Adsorption on porous hydrophobic supports is a very useful method for lipase immobilisation.
Entrapment Entrapment Entrapment in sol–gel materials. Entrapment in organic polymers.
Covalent coupling Covalent couplingCovalent coupling to a solid support is the most typical way of immobilising enzymes, and a variety of such methods has been described.There are several commercially available activated support materials intended for covalent enzyme immobilisation.
Cross-linking Cross-linkingTypical cross-linked preparations contain the enzyme as the main constituent, but cross-linking is also used in combination with other immobilisation methods, such as adsorption, to prevent enzyme leakage.
Cross-linked enzyme aggregatesCross-linked enzyme crystals
Surfactant-based lipase preparations
Surfactant-based lipase preparations
Surfactants can activate lipases in different immobilisation
procedures, probably by increasing the fraction of the open,
active lipase conformation. In addition, surfactants can be
used as the main agent for the preparation of lipases for use
in organic media.
Ion-paired lipasesSurfactant-coated lipasesLipases in microemulsionsMicroemulsion-based organogels
These methods can be classified into:
Water dependence of lipase-catalysedreactions
Water dependence of lipase-catalysedreactions
Water is always present, even when using organic solvents
as reaction media for lipase-catalysed reactions.
It was observed that the catalytic activity of enzymes
correlated well with the amount of water bound to the enzyme.
There is a large variation among the enzymes concerning
how the water in organic media influences their catalytic
activity.
Water dependence of lipase-catalysedreactions
Water dependence of lipase-catalysedreactions
Water has both positive and negative effects on the rate of lipase-catalysed reactions.
Positive effects of water•General activation due to increased internal flexibility of the enzyme•Increased active site polarity•Increased proton conductivity•Functions as substrate (increases hydrolysis only)
Negative effects of water•Inhibition (interference with substrate binding)•Formation of a diffusion barrier for hydrophobic substrates•Causes hydrolysis which competes with the desired reaction (esterification, transesterification)
Applications involving lipase-catalysedreactions
Applications involving lipase-catalysedreactions
• Ester synthesisThe majority of applications of lipases in organic media are in the preparation of esters, and a variety of esters has indeed been prepared.
• TriacylglycerolsStructured lipids, Interesterification of triacylglycerols
Lipases are excellent tools for the synthesis of triacylglycerols or the modification of existing triacylglycerols in organic media.
Applications involving lipase-catalysedreactions
Applications involving lipase-catalysedreactions
• Enantiomer resolutionThe lipase converts one of the enantiomers into a product that can easily be separated from the unreacted isomer.
• Fatty-acid enrichmentLipases can also be used for the enrichment of other isomers or closely related substances, provided that they have the appropriate substrate specificity.
• BiodieselTriacylglycerols are the normal substrates, lipases express high activity in biodiesel production.
• Phospholipid conversion• Carbohydrate modification• Increasing the lipophilicity of bioactive compounds
In which applications are lipases attractive?
In which applications are lipases attractive?
Lipase-catalysed reactions have proven to be better than other
approaches for the preparation of many products. The most
thoroughly studied applications are those that have been realised on
an industrial scale.
The regioselectivity of lipases makes them the natural choice as catalysts for the production of structured lipids. The mild reaction conditions which lead to fewer side reactions, higher product yields and less waste. Product purification is simplified by the reduction in by-products. Less environmental impact than alternative methods, which is naturally desirable.
Which is the best method of lipase immobilisation?
Which is the best method of lipase immobilisation?
The following are important in obtaining an immobilised lipase preparation that uses the enzyme as efficiently as possible.No enzyme inactivation should occur during immobilisationNo enzyme leakage should occur after immobilisationBe present in fully activated formMass transfer limitations should be negligible
The first two are sometimes difficult to combine. Covalent coupling to a support or covalent cross-linking are the best ways to avoid enzyme leakage, but they often cause enzyme inactivation.
Non-covalent immobilisation is sufficient because the lipase is not soluble in the reaction medium, but remains in the immobilised preparation.
The best approach may be not to immobilise the lipase, but to use surfactants to form hydrophobic ion pairs, surfactant-coated lipases or microemulsions. However, in these cases there is a need for a more sophisticated separation step to separate the enzyme from the product mixture.
Thank you for your attention
谢谢!
Thank you for your attention
谢谢!